IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-12

Tandem OriT by Mingzhi Qu, Ze Ren

colony PCR Test for pSC101 Conjugation Test(II)

• Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
• Test plate:LB- R751, Lb- pSB101, R751-pSC101 X Dh5α-R0040, R751 X Dh5α-R0040
• primer :R751 OriT primer, pSC101 primer.
• according to <Colony PCR STANDARD PROTOCOL>
• PCR system contains(each well):

0.5  µl      Primer 1(100uM)
0.5  µl      Primer 2
2    µl      dNTP(2.5uM)
2.5  µl      10X Taq Buffer
0.25 µl      Taq
19   µl      dH20
1    µl      template
--------------------------
~25  µl      Total

• PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
• PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .

electrophorsis result

• top:from left to right
  • 1-5 R751-pSC101 X Dh5α-R0040
  • 6-10 R751 X Dh5α-R0040
  • 11 R751
  • 13 Maker(DL2000 plus)
• botton:from left to right
  • 1-4 R751
  • 5-10 psc101
  • 11 Maker(DL2000 plus)

oriT Knock Out by Liu Ting

colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment

PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)

• PCR system for DH5a-oriT-deleted R751

pKO3-R(100uM)  2  ul
R1S(20uM)      10 ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          163ul
(template)
--------------------
Total          200ul

• PCR system for DH5a-oriT-deleted F

F1S(20uM)      10 ul
F2A(20uM)      10 ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          155ul
(template)
--------------------
Total          200ul

• PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment

S1S(100uM)     2  ul
S2A(100uM)     2  ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          171ul
(template)
--------------------
Total          200ul

PCR condition

for all the above three: 94℃ 10min; (94℃ 45s,51℃ 1min,72℃ 1min30s)/cycle X 30 cycles; 72℃ 10min; 16℃ 10min; end.

electrophorsis result

none...

Lock & Key By Yu Tao

Transformation: R0040<-J01010 and R0010<-J01008

Transformation Result: R0040<-J01010 and R0010<-J01008

• There are colonies both in the experimental plate and the negative control plate.
• For R0040<-J01010, more clonies grow in the expeimental plate, but the opposite result for R0010<-J01008.
• Number of colonies: around 100 or 200.
• Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.

Preparation of Competent Cells

• Because the newly produced competent cells are of low efficiency, I decide to make some competent cells once more.
• Transform 1uL B0015 competent cells for efficiency test.